In continuation of work on the nuclear hormone receptor (NCR) CHR3, we were able to show that a new technique in which RANI is generated in the bacteria which the worms ingest is more reliable than soaking worms in RANI. The gene of interest is introduced into the bacteria with two T7 RNA polymerase promoters . IPTG will induce the gene of interest and the worms are fed this construct. We how have tens of thousands of worms which are synchronized so we can estimate the activity of genes which might be induced or repressed by CHR3 at two hourly intervals between L1 and L2. We have primers which we have shown to work for eight candidate genes so with RT PCR we should be able to answer these questions in a relatively few weeks. Additionally, we performed several molting screens and found a single mutation on chromosome 3 near the dpy-18 locus which reproduced the results of CHR3 inhibition by its RANI.. We are currently attempting to isolate this gene. (M Kostrouchova, M. Krause)Previous work on c intestinalis identified allowed us to identify and sequence a nCR gene analogous to the TR. It did not bind T3 and we have been trying to clone additional genes but have been hampered by a lack of good DNA and RNA from cina. Hence for the instant we have shifted to studying aldehyde dehydrogenase genes in the scallop since this protein makes up all of the scallop lens protein. The gene has been isolated and sequenced (Piatigorsky, Carosa) Now we have examined the activity of the promoter of this gene in mouse lens cells, L929 mouse fibroblasts, and Cos 7 monkey kidney cells. The +63/-2120 construct hooked up to the luciferase gene was active in both mouse cell lines but inactive in the Cos 7 cells. There is a CREB like site in the scallop ALDH promoter and each of five nucleotides in it were mutated. All except one of these single base pair mutations abolished activity. One mutation which actually created a sequence identical to the mouse alph a crystalline promoter reduced but did not abolish activity. Three mutations were introduced into the alpha a CRYBP 1 site found in mouse and scallop lens promoters. None of the mutations destroyed activity and one that recreated the mouse site, increased activity two fold. Mutations in an AP1 site did not affect promoter activity. Gel shift assays showed specific protein binding to the scallop CREB site sequence from nuclear extracts of the alpha TN4-1 mouse lens cells or the L929 mouse fibroblast cells. No binding was seen with COS 7 cell nuclear extracts. Some binding was seen with scallop stomach and gill cells--lens cells are present in too small a quantity for adequate nuclear extracts. There is also a putative PAX 6 sequence in the scallop promoter and double transfection experiments showed that with a PAX 6 vector, activity of the scallop promoter could be doubled. Analysis of the sequences of important regions in thirteen well studied promoters of different lens crystalline genes from eight different species failed to identify a common motif. Thus lens specificity might be a function of a specific combination of multiple common sequences or, more likely, greater message and protein stability in the lens. (Carosa and Piatigorsky)We have continued work on nhrs from trepedilia cystophora and have shown that transfection of the jRXR gene into lens cells enhanced the activity of the J1B promoter (of the lens crystalline in that species) attached to firefly lucifers as a reporter. Both 9-is and all-trans resinoid acid enhanced further the activity of jRXR. This is somewhat curious since we have previously shown that jRXR specifically binds with high affinity only the is isomer. There was no result if 3T3 cells, P19 embryonal carcinoma, or Cos-7 cells were used. We have continued work on growing cystophora so they may metamorphose. We then plan to study gene expression during development using the yeast two hybrid screen. (Kostrouch, Z.)